Team:JNFLS/Protocols

Protocols

PCR truncation of HCVC gene

Primers:

tu1,2,3

Enzyme digestion of pcoldII vector plasmid

tu4

  1. Enzyme digestion of pcoldII vector plasmid
  2. add 5ul green buffer
  3. add 1ul Xhol
  4. add 1ul PstI
  5. add distilled water to 20μL
  6. water bath at 37 degrees for 10 min

    7. Ligation

    tu5

    Bacterial transformation

    1. Remove cells from -80°C freezer and thaw in hand
    2. Add 1-5 μl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex
    3. Place the mixture on ice for 2 minutes. Do not mix
    4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
    5. Place on ice for 2 minutes. Do not mix.
    6. Pipette 250 μl of room temperature SOC into the mixture. Immediately spread 50-100 μl onto a selection plate and incubate overnight at 37-42°C. NOTE: Selection using antibiotics other than ampicillin may require some outgrowth before plating on selective media. Colonies develop faster at temperatures above 37°C, however some constructs may be unstable at elevated temperatures.

      7. Colony PCR

      tu6

      Conditions：

      tu7

      Gel Recycling

      1. Run electrophoresis separation of the target DNA fragment
      2. Cut off the target DNA fragments, as far as possible the unwanted cut off
      3. The cut of the glue into the 1.5m | plastic centrifuge tube, said the quality of the plastic
      4. Add the same amount of Binding Buffer(XP2), that is, add 0.3m| liquid if 0.3g is weighed
      5. Heat in a 50-60 degree bath for a few minutes until all the glue is melted, stirring to mix.
      6. Put the HiBind DNA Mini Column into the 2ml collection tube
      7. Add the HiBindDNA Mini Column with less than 700ul from the DNA solution in step 5
      8. Centrifuge 60s at 12000rpm at room temperature
      9. Discard the waste liquid and recycle the collection pipe
      10. Repeat steps 7-9 until all samples are transferred to column
      11. Add 300ul Binding Buffer
      12. Centrifuge 60s at 12000rpm at room temperature
      13. Discard the waste liquid and recycle the collection pipe
      14. Add the 700ul SPW Wash Buffer
      15. Centrifuge 60s at 12000rpm at room temperature
      16. Discard the waste liquid and recycle the collection pipe once again in steps 14-16
      17. Centrifuge the empty HiBind DNA Mini Column 1 2000rmp for 2 minutes
      18. The Hibind DNA Mini Column was transferred to 1.5m| plastic centrifuge tube
      19. Add 15-30ul Elution Buffer to the center of the membrane
      20. Let sit at room temperature for 2 minutes
      21. Centrifugation for 12000rmp at room temperature for 1 minute